WO2017117467A1 - Hydrogels biocompatibles et conducteurs présentant des propriétés physiques et électriques régulables - Google Patents

Hydrogels biocompatibles et conducteurs présentant des propriétés physiques et électriques régulables Download PDF

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WO2017117467A1
WO2017117467A1 PCT/US2016/069340 US2016069340W WO2017117467A1 WO 2017117467 A1 WO2017117467 A1 WO 2017117467A1 US 2016069340 W US2016069340 W US 2016069340W WO 2017117467 A1 WO2017117467 A1 WO 2017117467A1
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bio
hydrogel
hydrogels
polymer
cell
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Nasim Annabi
Iman Noshadi
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Northeastern University
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F265/00—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00
    • C08F265/04—Macromolecular compounds obtained by polymerising monomers on to polymers of unsaturated monocarboxylic acids or derivatives thereof as defined in group C08F20/00 on to polymers of esters
    • C08F265/06—Polymerisation of acrylate or methacrylate esters on to polymers thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
    • C08H1/00—Macromolecular products derived from proteins
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/04—Alginic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/08—Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L67/00—Compositions of polyesters obtained by reactions forming a carboxylic ester link in the main chain; Compositions of derivatives of such polymers
    • C08L67/04—Polyesters derived from hydroxycarboxylic acids, e.g. lactones
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01B—CABLES; CONDUCTORS; INSULATORS; SELECTION OF MATERIALS FOR THEIR CONDUCTIVE, INSULATING OR DIELECTRIC PROPERTIES
    • H01B1/00—Conductors or conductive bodies characterised by the conductive materials; Selection of materials as conductors
    • H01B1/06—Conductors or conductive bodies characterised by the conductive materials; Selection of materials as conductors mainly consisting of other non-metallic substances
    • H01B1/12—Conductors or conductive bodies characterised by the conductive materials; Selection of materials as conductors mainly consisting of other non-metallic substances organic substances
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/34—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate
    • C08F220/36—Esters containing nitrogen, e.g. N,N-dimethylaminoethyl (meth)acrylate containing oxygen in addition to the carboxy oxygen, e.g. 2-N-morpholinoethyl (meth)acrylate or 2-isocyanatoethyl (meth)acrylate
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F251/00—Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F289/00—Macromolecular compounds obtained by polymerising monomers on to macromolecular compounds not provided for in groups C08F251/00 - C08F287/00
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2210/00—Compositions for preparing hydrogels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L51/00—Compositions of graft polymers in which the grafted component is obtained by reactions only involving carbon-to-carbon unsaturated bonds; Compositions of derivatives of such polymers
    • C08L51/02—Compositions of graft polymers in which the grafted component is obtained by reactions only involving carbon-to-carbon unsaturated bonds; Compositions of derivatives of such polymers grafted on to polysaccharides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L51/00—Compositions of graft polymers in which the grafted component is obtained by reactions only involving carbon-to-carbon unsaturated bonds; Compositions of derivatives of such polymers
    • C08L51/08—Compositions of graft polymers in which the grafted component is obtained by reactions only involving carbon-to-carbon unsaturated bonds; Compositions of derivatives of such polymers grafted on to macromolecular compounds obtained otherwise than by reactions only involving unsaturated carbon-to-carbon bonds

Definitions

  • Hydrogels are three-dimensional polymeric networks made from highly hydrophilic natural or synthetic monomers rendered insoluble by virtual, electrostatic, or covalent crosslinking (Guiseppi-Elie, A., 2010, Biomaterials, 31, 2701). They are widely used in biomedicine due to their close resemblance to the extracellular matrix (ECM), biocompatibility, and their tunable mechanical and biochemical properties (Yue, K., et al., 2015, Biomaterials, 73, 254; Ullah, F. et al., 2015, Mater Sci Eng C Mater Biol Appl, 57, 414).
  • ECM extracellular matrix
  • hydrogels are typically non-conductive, which limits their application as bioactive scaffolds for excitable cells such as nerve and muscle cells (Wu, Y. et al., 2016, Acta Biomaterialia, 33, 122).
  • Electrically conductive hydrogels are a class of new generation smart biomaterials that allow direct delivery of electrical, electrochemical, and electromechanical stimulation.
  • ECHs possess (i) high electrical conductivity, (ii) properties typical of hydrogels, namely, high degree of hydration, swellability, biocompatibility, and diffusivity of small molecules, as well as (iii) optical and electrochemical properties of their electroactive components (Guiseppi-Elie, A. 2010, Biomaterials, 31, 2701).
  • ECHs can be used as temporary scaffolds that enable adhesion, proliferation, migration, and differentiation of different cell types as well as electroactive modulation of neurons, cardiomyocytes, fibroblasts, preosteoblasts, endothelial cells, and mesenchymal stem cells (Baheiraei, N.
  • ECHs Biophysical properties of ECHs such as Young's modulus and elasticity influence tissue function, biodegradability, and local cell behavior in vivo (Xu, S. et al., 2016, Appl Biomater, 104, 640; and Engler, A. J. et al., Cell 2006, 126, 677).
  • water uptake and swellability of a hydrogel can impair its conductivity due to percolation of fluid through it (Baei, P. et al., 2016, Mater Sci Eng C Mater Biol Appl, 63, 131).
  • hydrogels include incorporation of (a) different base polymers, (b) bioactive components, (c) porogens, and (d) hydrophilic or hydrophobic moieties, and varying the degree of crosslinking between the polymer networks constituting the hydrogels (Moreira, L. S., et al., 2014, Int Orthop, 38, 1861; Kotanen, C. N. et al., 2013, Biomaterials, 34, 6318; Mawad, D. et al., 2012, Advanced Functional Materials, 22, 2692).
  • Biomaterials have been made conductive through the addition of nanomaterials (e.g. silver nanowires, gold nanoparticles, carbon nanotubes (CNTs), and graphene oxide) or conductive polymers (e.g. polyaniline, polypyrole, polythiophene) to polymeric matrices.
  • nanomaterials e.g. silver nanowires, gold nanoparticles, carbon nanotubes (CNTs), and graphene oxide
  • conductive polymers e.g. polyaniline, polypyrole, polythiophene
  • Bio-ILs room temperature ionic liquids
  • Bio-ILs are low melting organic salts having the following characteristics: low volatility, high ionic conductivity and electrochemical stability, excellent dissolution capability, and low toxicity (Vijayaraghavan, R. et al., 2010, Chem Commun, 46, 294; and Fukaya, Y. et al., 2007, Green Chem, 9, 1155).
  • Bio-ILs have been used as biocompatible and biodegradable materials for various applications such as cancer therapy, multi -responsive drug delivery, sensors, batteries, and biomedical implants.
  • Choline is a precursor for phospholipids (i.e. phosphatidylcholine and sphingomyelin), the main components of biological membranes. It is produced in human and plant tissues in significant amounts. Furthermore, choline is degraded into smaller molecules by certain microorganisms (Klein, R. et al., 2013, Rsc Adv, 3, 23347). As such, choline and its derivatives are physiologically and environmentally non-toxic.
  • cytotoxicity of a class of choline phosphate ionic liquid on a mammalian cell showed that choline based Bio-ILs were less cytotoxic than their counterparts in which sodium instead of choline was used as a counter ion (Klein, R. et al., 2013, Rsc Adv, 3, 23347).
  • Use of choline based salts and ionic liquids as non-toxic components of various compositions has been described in the following patent/patent applications: DE102009026598; WO 2010/023490; and WO 2010/078300.
  • a biocompatible and implantable battery containing a polymeric electrolyte blend having choline based Bio-ILs embedded in chitosan has also been reported (Jia, X. et al., 2014, ACS Appl Mater Interfaces, 6, 21110).
  • the resulting polymer blend was shown to be mechanically robust and highly conductive (8.9 ⁇ 10 "3 S/cm).
  • the conductive polymeric blend was shown to drive low power implanted medical devices, such as a cardiac pacemaker.
  • conjugated polymers Yet another group of materials that has attracted significant interest for biomedical applications is conjugated polymers.
  • Conjugated polymers have been investigated for use, among others, in tissue engineering (Garner, B. et al., 1999, Journal of Biomedical Materials Research, 44, 121; Schmidt, C. E. et al., 1997, Proc Natl Acad Sci USA, 94, 8948), wound healing (Collier, J. H. et al., 2000, J Biomed Mater Res, 50, 574), biofuel cells (Kim, J. et al., 2006, Biotechnol Adv, 24, 296), flexible electronics (Green, R. A. et al., 2009, Biomaterials, 30, 3637; Abidian, M. R.
  • conjugated polymers have been reported (Cao, B. et al., 2015, Chem Sci, 6, 782).
  • the backbone of the polymer provides electrical conductivity and the zwitterionic side chains impart biocompatibility, sensitivity to environmental stimuli, and controllable anti- fouling properties.
  • the zwitterionic polymer could not be degraded in vitro or in vivo.
  • one of the main requisites of hydrogels suitable for use in tissue engineering and drug delivery applications is that they should be able to undergo degradation, which could prevent damage to the surrounding tissues (Dong, D. Y. et al., 2016, Acs Appl Mater Inter, 8, 4442).
  • the present invention provides a new class of polymer-based biomaterials (i.e., electrically conductive hydrogels or ECHs) prepared by conjugating a biocompatible polymer with a bio-ionic liquid (Bio-IL) or a component thereof.
  • Biomaterials thus prepared are intrinsically conductive, and there is no need for adding an electroactive component such carbon nanotubes during their preparation. Their conductivity is tunable by adjusting factors such as the ratio of the bio-ionic liquid component to the polymer.
  • the materials are biocompatible and biodegradable. Methods of preparing these materials are also provided.
  • a “bio-ionic liquid” as used herein refers to a salt that has a melting temperature below room temperature (e.g., the melting temperature is less than 10°C, less than 15°C, less than 20°C, less than 25°C, less than 30°C, or less than 35°C) and that contains a cation and an anion, at least one of which is a biomolecule (i.e., a molecule found in a living organism) or a biocompatible organic molecule.
  • bio-ionic liquids are organic salts of choline, such as carboxylate salts of choline, choline bicarbonate, choline maleate, choline succinate, and choline propionate.
  • An ionic constituent of a bio-ionic liquid is a cation or anion component of a bio-ionic liquid.
  • ionic constituents of bio-ionic liquids for use in the invention are biocompatible organic cations such as choline and other biocompatible quaternary organic amines, as well as biocompatible organic anions such as carboxylic acids, including formate, acetate, propionate, butyrate, malate, succinate, citrate, and the like.
  • biocompatible polymer refers to an organic polymer found in a living organism or compatible with a living organism.
  • the polymer can be naturally occurring or synthetic and charged or uncharged.
  • the polymer is sufficiently hydrophilic to be capable of forming a hydrogel or serving as a component of a hydrogel.
  • biocompatible polymers for use in the invention include gelatin, elastin, elastin like polypeptides (ELP), chitosan, tropoelastin, collagen, hyaluronic acid (HA), alginate, poly(glycerol sebacate) (PGS), poly(ethylene glycol) (PEG), and poly(lactic acid) (PLA).
  • a biocompatible polymer, conjugate, or other molecule or composition is capable of being in contact with cells without compromising their viability, such as by causing cell death, inhibition of cell proliferation, or exhibiting toxic effects on cellular metabolism or physiology of the organism.
  • a hydrogel is biocompatible if cells applied on its surface or embedded within its matrix remain viable as measured over a period of days, e.g., 5 days, 10 days, or 30 days.
  • a "biodegradable" polymer, conjugate, or hydrogel of the invention is prone to being degraded within an organism within a short time, such as within 5, 10, 30, 60, or 100 days. Biodegradation is mediated by the action of cells and enzymes found within the organism in which the biodegradable material is implanted, and results in the chemical breakdown of the material into smaller molecules and their eventual removal from the organism.
  • One aspect of the invention is a biocompatible and preferably biodegradable hydrogel of tunable conductivity.
  • the hydrogel includes a biocompatible polymer conjugated to an ionic constituent of a bio-ionic liquid via a linker.
  • the linker is a chemical moiety that covalently binds the constituent of a bio-organic liquid to the biocompatible polymer and is biocompatible and preferably biodegradable. Suitable linkers include diacrylates, disulfides, and esters.
  • Embodiments of the above hydrogel can include one or more of the following features.
  • the ionic constituent of a bio-ionic liquid can be, for example, choline or another quaternary amine.
  • the ionic constituent is another cationic constituent of a bio-ionic liquid.
  • the ionic constituent is an anionic constituent of a bio-ionic liquid.
  • the polymer can be any biocompatible polymer, such as a polymer found in a living organism, from which a conjugate is formed by the covalent attachment of an ionic constituent of a bio-organic liquid through a linker moiety.
  • the polymer can be gelatin, elastin, one or more elastin-like polypeptides (ELP), collagen (any type of collagen or a mixture thereof), hyaluronic acid (HA), alginate, poly(glycerol sebacate) (PGS), or poly(ethylene glycol) (PEG).
  • ELP elastin-like polypeptides
  • HA hyaluronic acid
  • PPS poly(glycerol sebacate)
  • PEG poly(ethylene glycol)
  • the conductivity of the hydrogel is at least about 3.0 x 10 "5 siemens/meter (S/m).
  • the conductivity of the hydrogel can be from about 3.0 x 10 "5 S/m to about 1.3 x 10 "2 S/m.
  • the ratio of the polymer to the ionic constituent can range, for example, from 100:0 to 1 :4 by weight; i.e., the weight percentage of the ionic constituent of a bio-ionic liquid can range from 0 (or a small value > 0, e.g., 0.1) to about 80.
  • the conjugated polymer can be present at, for example, from 10% to 20% of the weight of the hydrogel, or from 11% to 20%, or 12% to 20%, or 15% to 20%, or about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% (all wt%).
  • the conjugated polymer can be present at from about 20 wt% to about 80 wt% of the hydrogel.
  • the conductivity may be tuned by changing the ratio of the polymer to the ionic constituent of the Bio-IL.
  • the conductivity may be tuned also by changing the percent weight of the total polymer in the hydrogel.
  • the compressive modulus of the hydrogel is between 0.60 ⁇ 0.20 kPa and 178.13 + 3.48 kPa.
  • the Young's modulus of the hydrogel is between 5.4 kPa and 100.77 + 3.48 kPa.
  • the compressive modulus and the Young's modulus of the hydrogel may be tuned by changing the ratio of the polymer to the Bio-IL.
  • the compressive modulus and the Young's modulus of the hydrogel may be tuned also by changing the percent weight of the total polymer in the hydrogel.
  • the porosity and the swellability of the hydrogel may be tuned by changing the ratio of the polymer to the Bio-IL or by changing the percent weight of the total polymer in the hydrogel.
  • the hydrogel is capable of supporting cell proliferation, organization, and/or function of an excitable cell in both 2D cell seeding and 3D cell encapsulation.
  • the excitable cell type for example, can be a nerve cell, a muscle cell, a fibroblast, a preosteoblast, an endothelial cell, or a mesenchymal stem cell.
  • the muscle cell is a cardiomyocyte.
  • Another aspect of the invention is a temporary scaffold for cells that supports electroactive modulation of the cells, the scaffold including the hydrogel according to the above-described aspect of the invention.
  • Embodiments of the above scaffold can have one or more of the following features.
  • the scaffold supports one or more of adhesion, proliferation, migration, and differentiation of the cells. These cells may be excitable cells, e.g., neurons, cardiomyocytes, fibroblasts, preosteoblasts, endothelial cells, or mesenchymal stem cells.
  • a method of preparing a conductive hydrogel includes: (a) providing an ionic constituent of a Bio-IL and a polymer, (b) modifying each of the ionic constituent and the polymer to generate a modified ionic constituent and a modified polymer, respectively, and (c) reacting the modified ionic constituent with the modified polymer using light-initiated polymerization to form the hydrogel.
  • the Bio-IL ionic constituent can be choline.
  • the polymer can be poly(ethylene) glycol.
  • the modified polymer can be poly(ethylene glycol) diacrylate.
  • the polymer can be gelatin.
  • the modified polymer can be gelatin acrylate (or methacrylate).
  • the light initiated polymerization can be carried out using Eosin Y, N- vinyl capro!actone (VC), and triethanolamine (TEOA), or Igracure (for UV) as photoinitiators.
  • the photoinitiator produces free radicals when exposed to ultraviolet (UV) or visi ble light.
  • photoinitiators examples include I-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-l- propane-l-one (Irgacure 2959, BASF, Florham Park, NJ, USA), azobi sisobutyronitrile, benzoyl peroxide, di-tert-butyl peroxide, 2,2-dimethoxy-2-pheny!acetophenone, Eosin Y, etc.
  • the photoinitiator is l-[4-(2-hydroxyethoxy)-ph.enyl]-2-hydroxy-2- methyl- 1 -propane- 1 -one.
  • the photoinitiator is Eosin Y.
  • the visible light activated photoinitiator is selected from the group consisting of: Eosin Y, triethanolamine, vinyl caprolactam, dl-2,3-diketo-l,7,7- trirnethylnorcamphane (CQ), 1 -phenyl-l,2-propadione (PPD), 2,4,6-trimethylbenzoyl- diphenylphosphine oxide (TPO), bis(2,6-dichlorobenzoyl)-(4-propylphenyl)phosphine oxide (Ir819), 4,4'-bis(dimethylamino)benzophenone, 4,4'-bis(diethylamino)benzophenone, 2- chlorothioxanthen-9-one, 4-(dimethylamino)benzophenone, phenanthrenequinone, ferrocene, diphenyl(2,4,6 trimethylbenzoyl)phosphine oxide/2-hydroxy-2-methylprop
  • the exemplary hydrogels and methods of the present invention provide several advantages. Hydrogels with different biomechanical and electroconductive profiles can be generated by varying the polymer to Bio-IL ratio and the concentration of the total Bio-IL conjugated polymer in the hydrogel. In other words, the biomechanical and electroconductive properties of the hydrogels are tunable. Further, the engineered hydrogels are biodegradable and elicit minimal inflammatory responses.
  • a biocompatible conductive hydrogel comprising a biocompatible polymer conjugated to a first ionic constituent of a bio-ionic liquid.
  • biocompatible polymer selected from the group consisting of gelatin, elastin, elastin like polypeptides (ELP), collagen, hyaluronic acid (HA), tropoelastin, chitosan, alginate, poly(glycerol sebacate) (PGS), poly(ethylene glycol) (PEG), and poly(lactic acid) (PL A).
  • Young's modulus of the hydrogel is from about 5 kPa to about 100 kPa.
  • hydrogel of any of the preceding embodiments, wherein the hydrogel is capable of supporting cell proliferation, tissue organization, and/or a function of an excitable cell.
  • the conductive hydrogel of any of the preceding embodiments that is biodegradable.
  • the hydrogel further comprises a second ionic constituent of a bio-ionic liquid, the second ionic constituent having a charge opposite to that of said first ionic constituent.
  • a method of preparing a conductive hydrogel including any of the conductive hydrogels of embodiments 1-21, the method comprising reacting an ionic component of a bio-ionic liquid with a biocompatible polymer to form the hydrogel; wherein the ionic component comprises a first functional group, the biocompatible polymer comprises two or more second functional groups, and the first and second functional groups react to form a linker that conjugates the biocompatible polymer to said ionic component.
  • biocompatible polymer is selected from the group consisting of gelatin, elastin, elastin like polypeptides (ELP), collagen, hyaluronic acid (HA), alginate, poly(glycerol sebacate) (PGS), and poly(ethylene glycol) (PEG).
  • a conductive hydrogel made by the method of any of embodiments 22-29.
  • a cell scaffold that enables electroactive modulation of cells bound to the scaffold comprising the hydrogel of any of embodiments 1-21.
  • the cell scaffold of embodiment 31 or 32, wherein the cells are selected from the group consisting of neurons, cardiomyocytes, fibroblasts, preosteoblasts, endothelial cells, mesenchymal stem cells, and combinations thereof.
  • the cell scaffold of embodiment 34, wherein the bound cells are selected from the group consisting of neurons, cardiomyocytes, fibroblasts, preosteoblasts, endothelial cells, mesenchymal stem cells, and combinations thereof.
  • FIGS. 1A-1E are diagrams showing synthesis and characterization of Bio-IL functionalized gelatin methacrylol (GelMA) hydrogel.
  • FIG. lA is a schematic diagram of the reaction for acrylation of Bio-IL (choline bicarbonate) to produce acrylated Bio-IL (choline acrylate).
  • FIG. IB is a schematic diagram of the reaction between GelMA and acrylated Bio-IL in the presence of eosin Y and visible light to form a GelMA-Bio-IL hydrogel.
  • FIGS. 1C-1E show 1 H-NMR spectra of choline acrylate (acrylated Bio-IL, FIG. 1C), gelatin methacrylate (FIG.
  • FIG. IE GelMA/Bio-IL hydrogel was formed by using 1% VC, 1.5% TEOA, and 0.1 mM Eosin Y at 120 sec light exposure).
  • FIG. 2A is a diagram showing the experimental setup of a two-probe electrical station used to measure electrical conductivity of the engineered GelMA-Bio-IL hydrogels.
  • FIGS. 2B and 2C are graphs showing measurements of conductivities of Bio-IL-conjugated GelMA and PEGDA polymers, respectively, at different polymer concentrations and polymer to Bio- IL ratios.
  • FIG. 2D is a graph showing electrical conductivity measurements of GelMA-Bio- IL hybrid hydrogels at 20%, 30%, and 40% stretch. Each hydrogel has a polymer concentration of 15% and a polymer to Bio-IL ratio of 50:50.
  • FIG. 2A is a diagram showing the experimental setup of a two-probe electrical station used to measure electrical conductivity of the engineered GelMA-Bio-IL hydrogels.
  • FIGS. 2B and 2C are graphs showing measurements of conductivities of Bio-IL-conjugated GelMA and PEGDA polymers, respectively, at different polymer concentrations and polymer to Bio-
  • FIG. 2E is a schematic diagram of ex vivo studies performed using rat abdominal muscle tissues connected using a GelMA:Bio-IL hydrogel having a final polymer concentration of 15% and a GelMA:Bio-IL ratio of 50:50 (bottom right) and pure GelMA hydrogel (bottom left).
  • FIG. 2F is a graph showing threshold voltages at which contraction was achieved using GelMA:Bio-IL hydrogels having a final polymer concentration of 15% and GelMA:Bio-IL ratios of 50:50 and 20:80 ratios as well as using pure GelM A hydrogels having a final polymer concentration of 15%.
  • FIGS. 3A-3D are graphs showing measurements of mechanical properties of Bio-IL conjugated GelMA and PEGDA hydrogels crosslinked with visible light. Crosslinking was carried out by exposure to light for 120 seconds in the presence of 1% VC, 1.44% TEOA, and 0.1 mM Eosin Y.
  • FIGS. 3 A and 3B are graphs showing compressive moduli of GelMA-Bio- IL and PEGDA-Bio-IL hydrogels, respectively, having varying polymer concentrations and polymer to Bio-IL ratios.
  • FIGS. 4A-4H are images and graphs showing in vitro degradation, swelling, and pore characteristics of polymer-Bio-IL hydrogels.
  • FIGS. 4A and 4B are representative scanning electron microscope (SEM) images of GelMA-Bio-IL and PEGDA-Bio-IL hydrogels, respectively, formed by using polymer to Bio-IL ratios of 100:0 (left) and 1 : 1 (right) at a polymer concentration of 15% (w/v).
  • FIGS. 4C and 4D are graphs showing average pore sizes of GelMA-Bio-IL and PEGDA-Bio-IL hydrogels at varying polymer concentrations and polymer to Bio-IL ratios.
  • FIGS. 4E and 4F are graphs showing swelling ratios of GelMA- Bio-IL and PEGDA-Bio-IL hydrogels, respectively, in DPBS (Dulbecco's phosphate buffered saline) at 4, 8, and 24 hours.
  • FIGS. 5A-5I are immunofluorescent images and graphs showing results of in vitro 2D culture of cardiomyocytes seeded on the surface of GelMA-Bio-IL hydrogels.
  • FIG. 5E is a graph showing viability levels of cardiomyocytes cultured on GelMA (control) and GelMA-Bio-IL hydrogels. Viability measurements were performed after 1, 3, and 5 days of culture.
  • FIG. 5F is a graph showing metabolic activity levels of the cardiomyocytes in RFU (relative fluorescence intensity) obtained using the PrestoBlue assay (Life Technologies). Metabolic activities were measured at 1, 3, and 5 days after seeding the hydrogels with cardiomyocytes.
  • FIGS. 6A-6K are a set of images and graphs showing in vivo biodegradation and biocompatibility of GelMA-Bio-IL hydrogels in a rat subcutaneous animal model.
  • FIG. 6B is a set of images of GelMA-Bio-IL hydrogels taken on days 0, 4, 14, and 28 post implantation.
  • FIGS. 6C, 6D, and 6E are immunofluorescence images showing in vivo degradation of GelMA-Bio-IL hydrogels.
  • FIGS. 7A-7D are a set of representative graphs showing evaluation of mechanical properties of Bio-IL functionalized GelMA and PEGDA hydrogels crosslinked with visible light.
  • FIGS. 7 A and 7B show representative compression stress curves for GelMA-Bio-IL and PEGDA-Bio-IL hydrogels, respectively, at polymer concentrations of 10%, 15%, and 20%) and a polymer to Bio-IL ratio of 80:20.
  • FIGS. 7C and 7D show representative tensile stress curves for GelMA-Bio-IL and PEGDA-Bio-IL hydrogels, respectively, at polymer concentrations of 10%>, 15%, and 20% and a polymer to Bio-IL ratio of 80:20. Error bars indicate standard error of the means.
  • FIGS. 8A-8F are a set of graphs showing the evaluation of in vitro degradation of Bio-IL functionalized GelMA hydrogels.
  • FIGS. 8A, 8B, and 8C are graphs showing degradation rates of GelMA-Bio-IL hydrogels having polymer concentrations of 10%, 15%, and 20%), respectively, in DPBS.
  • FIGS. 8D, 8E, and 8F are graphs showing degradation rates of GelMA-Bio-IL hydrogels as in FIGS. 8A, 8B, and 8C, respectively, except that the hydrogels were kept in a solution containing DPBS supplemented with 10%> FBS. For each of the polymer concentrations, hydrogels having four different ratios of GelMA and Bio-IL were tested. The tests were conducted over a 14 day period.
  • FIGS. 9A-9F are a set of graphs showing the evaluation of in vitro degradation of PEGDA-Bio-IL hydrogels.
  • FIGS. 9A, 9B, and 9C are graphs showing degradation rates of the hydrogels having polymer concentrations of 10%>, 15%, and 20%, respectively, in DPBS.
  • FIGS. 9D, 9E, and 9F are graphs showing degradation rates of PEGDA-Bio-IL hydrogels as in FIGS. (A), (B), and (C), respectively, except that the hydrogels were kept in a solution containing DPBS supplemented with 10%> FBS. For each polymer concentration, hydrogels having four different ratios of GelMA and Bio-IL were tested. The tests were conducted over a 14-day period.
  • FIG. 10A is an 1H MR spectrum of choline bicarbonate.
  • Hydrogels with electroconductive properties have potential for use as bioactive scaffolds in tissue engineering where growth and stimulation of excitable cells is required.
  • Conductive hydrogels are also widely used in biomedical applications such as electroactive drug delivery devices, biorecognition elements for implantable biosensors, and organic coatings for neural interfaces.
  • implantable hydrogels should be biocompatible and biodegradable such that they may be introduced into living organisms without eliciting inflammatory responses.
  • the present invention is generally directed towards conductive hydrogels that are biocompatible and biodegradable and possess tunable conductivity. More specifically, the invention provides a biodegradable and biocompatible hydrogel of tunable conductivity that includes a bio-ionic liquid (Bio-IL) conjugated to a polymer.
  • Bio-IL bio-ionic liquid
  • the bio- ionic liquid is choline.
  • the polymers are gelatin methacrylol (GelMA) (FIG. IB) and polyethylene glycol) diacrylate (PEGDA).
  • GelMA and PEGDA based polymer systems are intrinsically non-conductive, which limits their use in applications requiring modulation of excitable cells such as neurons and muscle cells.
  • Incorporation of Bio-IL into the polymer network provided tunable electroconductive properties to the Bio-IL conjugated hydrogels (engineered scaffolds).
  • Conductive hydrogels according to the present disclosure have a conductivity of at least 3.0 x 10 "5 siemens/meter (S/m). In one embodiment, the conductivity is as high as 1.3 x 10 "2 S/m.
  • Hydrogels according to the present disclosure may be made with varying ratios of polymer to Bio-IL.
  • the ratio of polymer to Bio-IL is from about 100:0 to about 20:80.
  • the conjugated polymer may be present at 10% to 20% of the weight of the hydrogel.
  • Hydrogels used in biomedical applications must provide adequate mechanical support to cells and tissues. They should be effective also in transducing physicochemical cues to the cells and tissues given that different mechanical cues are known to modulate key cellular functions such as cell proliferation, differentiation, migration, and apoptosis (Chicurel, M. E. et al., 1998, Curr Opin Cell Biol, 10, 232). Therefore, in order to reproduce the mechanical features of native tissues it is desirable that the engineered biomaterials have tunable physical properties.
  • the compressive modulus of the hydrogel described in the present disclosure ranges between 0.60 ⁇ 0.20 kPa and 178.13 + 3.48 kPa.
  • the Young's modulus ranges between 5.4 kPa and 100.77 + 3.48 kPa.
  • both the compressive modulus and the Young's modulus of the hydrogel may be tuned by changing the polymer to Bio-IL ratio. Both parameters may be tuned also by changing the percent weight of the conjugated polymer.
  • the porosity of hydrogels plays a major role in the modulation of cell and tissue interactions as well as in the penetration of cells into the scaffold in 2D culture and 3D encapsulation (Annabi, N. et al., 2010, Tissue Eng Part B Rev, 16, 371). Scaffolds with higher porosity are desirable for tissue engineering as they are better penetrated by cells and, as such, favor formation of new tissue within the 3D structure of the scaffold. Hydrogels with tunable porosity, therefore, are useful for generating cell-laden scaffolds with different spatial distributions (Annabi, N. et al., 2010, Tissue Eng Part B Rev, 16, 371; Zeltinger, J. et al., 2001, Tissue Eng, 7, 557).
  • porosity and swellability can be tuned by changing the ratio of the polymer to the bio-ionic liquid.
  • the porosity and the swellability of the hydrogel can be tuned also by changing the percent weight of the conjugated polymer.
  • the hydrogels described here are also capable of supporting proliferation, organization, and function of excitable cells.
  • Excitable cells include nerve cells, muscle cells (e.g., cardiomyocytes), fibroblasts, preosteoblasts, endothelial cells mesenchymal stem cells and some endocrine cells (e.g., insulin producing pancreatic ⁇ cells).
  • PDMS polydimethylsiloxane
  • TEOA triethanolamine
  • VC N-vinylcaprolactam
  • the PDMS material contained rectangular (w: 5 mm, ⁇ £: 12 mm, d: 1.25 mm) and cylinder-shaped molds (d: 5.5 mm, h: 4 mm) for conducting tensile and compression tests, respectively. Samples were removed from the molds and placed in DPBS for 2 hours at room temperature.
  • Hydrogels were blotted dry and measurements for swelling made using digital calipers before positioning them in an Instron 5542 mechanical tester with a 10 N load cell. Compression was performed at 1 mm/min of speed until failure occurred. Compression modulus was calculated as the slope of the initial linear region at the stress- strain curve obtained by plotting the results of compressions.
  • hydrogels were formed into rectangular shapes and fixed to fine adhesive tape. Each end of the adhesive tape was attached to the Instron and the sample was stretched at a rate of 2 mm/min until failure occurred. Elastic moduli were calculated by obtaining the slope of the stress-strain curves.
  • Hydrogels produced by using various ratios of polymer to Bio-IL as well as different polymer concentration were formed in a 70 ⁇ ⁇ rectangular PDMS mold and allowed to sit for 24 hours. Once dried, conductivity analysis was performed using a two-probe electrical station connected to a Hewlett Packard (HP) 4155 A Semiconductor Parameter analyzer. Each hydrogel was measured and placed in a relaxed state where the two probes penetrated the hydrogels - one at each end (FIG. 2C). The analyzer was set to measure current in the presence of an electrical stimulation ranging from -5 to 5 volts. Results were analyzed to determine conductivity values. In vitro degradation test
  • Freeze-dried samples of hydrogels were weighed and placed in a 24 well plate with 1 ml of DPBS or DPBS supplemented with 10% FBS at 37°C in a humidified oven for 2 weeks.
  • the DPBS/FBS solutions in the plate were replaced with fresh solutions every three days to maintain constant enzyme activity. At prearranged time points (after 1, 7, and 14 days), the samples were removed from the DPBS/FBS solutions, freeze-dried and weighed. Percentage degradation (D%) of the hydrogels was calculated using Equation (1): (1) where Wi is the initial dry weight of the sample and W t is the dry weight after time t.
  • the equilibrium swelling ratio of GelMA-Bio-IL and PEGDA-Bio-IL hydrogels were evaluated.
  • cylinder-shaped hydrogels were prepared (7 mm in diameter, 2 mm in depth). Prepared hydrogels were washed three times with DPBS. Next, they were lyophilized and weighed in dried condition. Thereafter, the samples were immersed in DPBS at 37 °C for 4, 8, and 24 hours and weighed again after immersion.
  • the swelling ratio and water uptake capacity of the samples were calculated as the ratio of the swelled sample mass to the mass of the lyopholized sample.
  • ICAUC Institutional Animal Care and Use Committee
  • atria and blood vessels were carefully removed and each heart was quartered and incubated overnight in a solution of 0.05% trypsin (w/v) in HBSS at 4 °C. Trypsin digestion was stopped by adding culture media, followed by shaking for 5 min at 37 °C in a water bath. The tissues were then serially digested in 0.1% collagenase type II solution in HBSS (10 min shaking incubation at 37 °C). The collagenase solution containing the cardiomyocytes was centrifuged at 500 x g for 5 min. Primary cells were resuspended in DMEM supplemented with 10% FBS and preplated for 1 h to enrich for cardiomyocytes.
  • precursor hydrogel solutions were prepared in cell culture medium containing TEA (1.8% w/v) and VC (1.25% w/v), and gently mixed with cells (10 x 106 cells/ml). A single 7 ⁇ drop of this mixture was pipetted on a 150 ⁇ spacer, and covered by a TMSPMA-coated glass slide. After photocrosslinking, the hydrogels were washed several times with warm medium to remove the unreacted photoinitiators. The cell- laden gels were then placed in 24 well plates and incubated at 37°C, 5% C0 2 , and humidified atmosphere.
  • GelMA-Bio-IL hydrogels was evaluated using a commercial live/dead cell viability kit (Invitrogen) according to instructions from the manufacturer. Briefly, cells were stained with 0.5 L/mL of calcein AM and 2 pIJmL of ethidium homodimer-1 (EthD-1) in DPBS for 15 min at 37 °C. Fluorescent image acquisition was carried out at days 1, 4, and 7 post-seeding using an AxioObserver Zl inverted microscope (Zeiss). Viable cells appeared green and apoptotic/dead cells appeared red. The number of live and dead cells was quantified using the ImageJ software. Cell viability was determined as the number of live cells divided by the total number of live and dead cells.
  • Metabolic activity was evaluated at days 1, 3, and 5 post-seeding using the PrestoBlue assay (Life Technologies) according to manufacturer's instructions. Briefly, 2D cultures of primary cardiomyocytes were incubated in 400 ⁇ _, of growth medium with 10% PrestoBlue reagent for 2 h at 37 °C. Resulting fluorescence was measured (excitation 530 nm; emission 590 nm) using a Synergy HT fluorescence plate reader (BioTek). Control wells without cells were used to determine background fluorescence for all experiments.
  • GelMA-Bio-IL hydrogels (1 x 5 mm disks) were implanted into the pockets followed by anatomical wound closure and recovery from anesthesia. Animals were euthanized by anesthesia/exsanguination at days 4, 14, and 28 post-implantation, after which the samples were retrieved with the associated tissue and placed in DPBS.
  • Histological analysis and immunofluore scent staining Histological analyses were performed on cryosections of explanted hydrogel samples in order to characterize the inflammatory response elicited by the implanted material. After explantation, samples were fixed in 4% paraformaldehyde for 4 hours, followed by overnight incubation in 30% sucrose at 4 °C. Samples were then embedded in Optimal Cutting Temperature compound (OCT) and flash frozen in liquid nitrogen. Frozen samples were then sectioned using a Leica Biosystems CM3050 S Research Cryostat. 15- ⁇ cryosections were obtained and mounted in positively charged slides using DPX mountant medium (Sigma).
  • OCT Optimal Cutting Temperature compound
  • the slides were processed for hematoxylin and eosin staining (Sigma) according to instructions from the manufacturer. Immunohistofluorescence staining was performed on mounted cryosections as previously reported (Annabi, N. et al., 2016, Adv Mater, 28, 40). Anti-CD3, anti-osterix (SP7) (abl6669), and anti-CD68 (abl25212) (Abeam) were used as primary antibodies. An Alexa Fluor 488 -conjugated secondary antibody (Invitrogen) was used for detection. All sections were counterstained with DAPI (Invitrogen) and visualized on an AxioOb server Zl inverted microscope (Zeiss).
  • Immunocytofluorescence staining was performed on 2D cultures of primary cardiomyocytess to evaluate the expression of the cardiac differentiation marker sarcomeric a-actinin. Briefly, 2D cultures growing on the surface of GelMA and GelMA-Bio-IL hydrogels were fixed in 4% paraformaldehyde for 1 h at room temperature at day 7 post- seeding. Samples were washed three times with DPBS, permeabilized in 0.1% (w/v) Triton X-100 for 30 min, and blocked in 10% (v/v) goat serum in PBS containing 0.1% Triton x-100 for 1 h.
  • Samples were incubated overnight with an anti-sarcomeric a-actinin primary antibody (1 :200 dilution) in 10% (v/v) goat serum at 4 °C. After incubation, samples were washed three times with DPBS and incubated for 2 h at room temperature with an Alexa Fluor 488-conjugated secondary antibody diluted in 10% (v/v) goat serum (1 :200 dilution). Lastly, the samples were washed three times with DPBS and counterstained with DAPI (1/1000 dilution in DPBS) for 5 min at room temperature. Image acquisition was performed using an Axi oOb server Zl inverted microscope.
  • FIGS. 1 A- I E A versatile method for preparing Bio ⁇ IL conjugated hydrogels was developed. The method requires conjugating choline based Bio-ILs with natural and synthetic polymers to yield new biodegradable and conductive biomaterials (see FIGS. 1 A- I E).
  • Gelatin methacryloyl was synthesized as previously described (Nichol, J. W. et al., 2010, Biomaterials, 31, 5536). Choline acrylate was synthesized by reacting choline bicarbonate with acrylic acid. See FIG. 1A. Different ratios of GelMA and acrylated Bio-IL were mixed at room temperature. The resulting GelMA/Bio-IL prepolymer was then crosslinked into a hydrogel via visible-light initiated photopolymerization, using Eosin Y, vinyl caprolactone (VC), and triethanoiamine (TEOA) (FIG. IB). Composite hydrogel s were synthesized using 100/0 (control), 80/20, 50/50, and 20/80 polymer/Bio-IL ratios, as well as 10%, 15% and 20% (w/v) final polymer concentrations.
  • the acrylation of choline bicarbonate was confirmed by comparing the NMR spectra of choline bicarbonate with that of the choline acrylate (Bio-IL) as shown in FIG.1 OA.
  • the appearance of a peak related to the hydrogen atoms in the acrylate groups at ⁇ 5.8-6.1 ppm was indicative of the acrylation of the Bio-IL (FIG. 10B).
  • the ! H NMR spectra were collected for GelMA (FIG. ID), and GelMA/Bio-IL conjugate (FIG. IE) to confirm the conjugation of Bio-IL to GelMA.
  • PEGDA-Bio-IL hydrogels at 10% final polymer concentration exhibited conductivities of 9.32 X 10 "4 S/m and 4.86 X 10 "3 S/m at polymer to Bio-IL ratios of 4: 1 and 1 : 1, respectively (see FIG. 2C).
  • the conductivities of PEGDA/Bio-IL hydrogels were calculated to be 1.45 X 10 "3 S/m, 6.08 X 10 " 3 S/m, and 7.11 x 10 "3 S/m at polymer to Bio-IL ratios of 4: 1, 1 : 1, and 1 :4, respectively.
  • PEGDA/Bio-IL hydrogels at 20% polymer concentration exhibited conductivities of 1.48 X 10 "3 S/m, 1.26 X 10 "2 S/m, and 9.83 X 10 "3 S/m at polymer to Bio-IL ratios of 4: 1, 1 : 1, and 1 :4 respectively.
  • FIG. 2E A schematic diagram of the experimental set up used for this purpose is shown in FIG. 2E. Briefly, the rectus abdominis muscles of female Wistar rats were explanted after euthanasia and cut into square pieces. The tissues were placed 3 mm apart, in an electrically insulated polydimethylsiloxane (PDMS) mold. GelMA-Bio-IL hydrogels as well as pure GelMA hydrogel (control) were photocrosslinked in situ between two pieces of tissue (FIG. 2E).
  • PDMS electrically insulated polydimethylsiloxane
  • Pulsed direct current test runs were conducted by applying 50 ms square pulses at increasing frequencies using short platinum wires that were positioned on one of the two pieces of muscle tissue. The induction of contraction was visually inspected in the sample on the opposite end of the hydrogel after applying electrical pulses at increasing voltages. Results demonstrated that muscle tissue samples joined together using GelMA-Bio-IL hydrogels exhibited a significantly lower excitation threshold when compared to pure GelMA hydrogel controls (FIG. 2F). These observations indicated that Bio-IL functionalized polymers could be used to restore functional integrity in tissues in which electrophysiological communication has been interrupted.
  • GelMA-Bio-IL hydrogels exhibited highly tunable compressive moduli in the range of 0.60 kPa to 32.07 kPa (see FIG. 3 A).
  • the compressive moduli were found to be 2.65 + 1.06 kPa, 9.2 + 5.27 kPa, and 6.50 + 2.38 kPa, at polymer to Bio-IL ratios of 1 : 1, 4: 1, and 1 :0, respectively.
  • the condition corresponding to 10% polymer concentration and 1 :4 polymer-Bio- IL ratio did not lead to structurally integral hydrogels, suggesting that 10% polymer concentration is below the threshold needed to allow for hydrogel formation.
  • the compressive moduli were found to be 0.60 + 0.20 kPa, 5.53 + 0.76 kPa, 15.43 + 0.40 kPa, and 22.10 + 1.55 kPa, at polymer to Bio-IL ratios of 1 :4, 1 : 1, 4: 1, and 1 :0, respectively.
  • the compressive moduli were found to be 1.27 + 0.30 kPa, 28.73 + 0.55 kPa, 82.00 + 0.80 kPa, and 78.17 + 4.87 kPa at polymer to Bio-IL ratios of 1 :4, 1 : 1, 4: 1, and 1 :0, respectively.
  • GelMA-Bio-IL also exhibited highly tunable Young's moduli in the range of 5.40 kPa to 100.77 kPa, (FIG. 3C). Similar to compressive modulus, the values for Young's modulus also increased at higher polymer concentration values. For GelMA-Bio-IL hydrogels corresponding to 10% polymer concentration, the Young's moduli were found to be 5.20 + 1.15 kPa, 29.33 + 9.65 kPa, and 38.90 + 33.18 kPa at polymer to Bio-IL ratios of 1 : 1, 4: 1 and 1 :0, respectively. See FIG. 3C.
  • PEGDA-Bio-IL hydrogels at 20% CPC yielded Young's moduli of 7.70 + 1.58 kPa, 54.37 + 6.34 kPa, 125.33 + 3.56 kPa, and 172.70 + 2.86 kPa at polymer to Bio-IL ratios of 1 :4, 1 : 1, 4: 1, and 1 :0, respectively.
  • GelMA-Bio-IL hydrogel was tested for its ability to support cell proliferation, organization, and function in vitro.
  • Primary rat cardiomyocytes were used as exemplary excitable cells for these tests.
  • Commercially available assays were used with cardiomyocytes growing on the surface of GelMA or GelMA-Bio-IL over a period of 5 days to quantify live versus dead cardiomyocytes and to assess their metabolic activity to determine cell viability. See FIGS. 5A to 5D.
  • FIGS. 5E to 5H See FIGS. 5E to 5H.
  • Cardiomyocytes maintained in 2D environments tend to revert to a less mature phenotype and lose the ability to respond to physiologic stimuli. Hence, in addition to maintaining a metabolically active state, preservation of native phenotype is critical to promote spatial and functional organization of cardiomyocytes.
  • Immunofluorescence staining of the cardiac differentiation marker sarcomeric a-actinin revealed that cardiomyocytes in the GelMA-Bio-IL hydrogels were distributed in spatially-relevant multicellular organization (FIG. 5, (i) and (j)). These images also show that cardiomyocytes seeded on the surface of the engineered hydrogels exhibit cross-striations, which are indicative of the sarcomeric structures present in the native ventricular myocardium.
  • Biodegradable and biocompatible electroconductive scaffolds with varying degrees of biodegradability may be prepared by conjugating bio-ionic liquid to different polymers with varying degrees of stability.
  • biomaterials with more hydrolytically-stable backbones such as PEG diacrylamide (PEGDAA) (Browning, M. B. et al., 2014, J Biomed Mater Res A, 102, 4244.) would be ideal for long-term applications where a more biostable implantable scaffold is needed.
  • PEG diacrylamide PEGDAA
  • the biodegradability profile of GelMA-Bio-IL hydrogels allows for sustained cellular ingrowth as well as the eventual replacement of the implanted sample with new autologous tissue. Histological assessment of explanted hydrogels revealed ingrowth of predominantly non-inflammatory tissue and low deposition of fibrous collagenous capsule. See FIGS. 6A- 6C. This observation was further confirmed by immunohistofluorescence analysis of the explanted samples. Fluorescence immunostaining of CD3 and CD63 antigens showed no sustained infiltration by pro-inflammatory leukocytes (see FIGS. 6F-6H) and macrophages (see FIG. 6I-6K), respectively. The results indicate that GelMA-Bio-IL hydrogels elicit minimal inflammatory responses when implanted subcutaneously in an animal host.
  • Scaffolds made with a given biopolymer may be particularly suitable for a certain type of physiological response.
  • previous studies have demonstrated the suitability of GelMA-based hydrogels for the induction of angiogenesis (Dreesmann, L. et al., 2007, Biomaterials, 28, 5536).
  • Bio-IL-conjugation of different bioactive polymers such as alginate could be used for studies involving osteogenesis as well as other bone tissue engineering applications (Xia, Y. et al., 2012, Journal of Biomedical Materials Research Part A, 100a, 1044).
  • a biomaterials-based approach like the one presented in this invention can help restore ventricular function by mechanically and electrically coupling the area around the infarcted myocardium. Furthermore, in addition to its role in excitation-contraction coupling, electrical stimulation of cardiomyocytes is also known to modulate cell proliferation and function through the calcium/calmodulin pathway (Titushkin, I, et al., Tissue Eng Part B Rev 2013, 19, 48.). The ability of the hybrid GelMA-Bio-IL hydrogels described herein to efficiently transduce multiple physiological stimuli to modulate tissue function holds great potential for use in cardiac tissue engineering applications. All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

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Abstract

L'invention concerne un hydrogel biodégradable et biocompatible de conductivité régulable. L'hydrogel comprend un polymère conjugué à un liquide bio-ionique. Les propriétés mécaniques et électriques de l'hydrogel peuvent être variées par la modification du rapport du polymère au liquide bio-ionique dans le polymère conjugué. Ces propriétés peuvent également être variées par modification du pourcentage en poids du polymère conjugué dans l'hydrogel. L'invention concerne également un procédé de préparation de l'hydrogel.
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WO2021124225A1 (fr) * 2019-12-18 2021-06-24 Allergan Pharmaceuticals International, Ltd Matériaux polymères hybrides et leurs utilisations
CN115605237A (zh) * 2019-12-18 2023-01-13 阿勒根制药国际有限公司（Ie） 杂化聚合物材料及其用途

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